This application generally relates to the area of optical microscopy, and specifically to patterned illumination in optical microscopy. Optical microscopy can be used in a variety of applications, including for viewing of biological samples and imaging of metallurgical objects. Optical widefield microscopy refers to broad illumination of a specimen in the object plane and obtaining images therefrom, in contrast to scanning confocal microscopy which refers to illumination and imaging of pin-hole sections and using recombining methods to reproduce the image. Optical widefield microscopy provides some visual depth in the axial direction (z-plane).
In optical microscopy, photobleaching is used to refer to the fluorescent excitation and resultant photochemical destruction of a fluorophore when under fluorescent illumination. For example, a fluorophore may be used in a fluorescently labeled or tagged substance, molecule or protein in a biological specimen, wherein the fluorescence emission is then projected to the image detector plane. However, since photobleaching occurs, this may complicate the observation of the fluorescent molecules, since they will eventually be destroyed by the light exposure necessary to stimulate them into fluorescing. This may be especially problematic in time-lapsed microscopy.
Fluorescence recovery after photobleaching (FRAP) refers to an optical technique which takes advantage of the principle of photobleaching to provide both qualitative and quantitative information about fluorescently labeled substances, molecules or proteins in a biological specimen. In FRAP, only a specified region of the biological specimen is illuminated, using for example a narrow bar pattern, until all or most fluorescence in the region of interest is bleached, then the movement or mobility of fluorescently labeled entities into this photobleached zone are monitored to observe preferred sites of localization of the new fluorescently labeled molecules. The fluorescence recovery in the photobleached zone may also be quantified to provide diffusion coefficients for the molecules of interest.
Photoactivation refers to an optical technique characterized by increased fluorescence contrast when a short pulse of activating light is used to photo-activate activatable molecules or proteins. In photoactivation, there is also only a specified region of the biological specimen illuminated and the movement or mobility of the activated fluorescently labeled entities is monitored and quantified.
In some conventional microscopes, the optical components assembled within are often assembled at the manufacturer level to provide rigid and exact positioning and orientations. In some attempts to improve resolution, complex configurations of lenses, light sources, and relatively expensive lasers may also be assembled by the manufacturer. Such microscopes may not provide the desired flexibility in substitution of components, or upgrading when a better-resolution component becomes available.
For example, in some applications a grid, Ronchi Ruling or diaphragm is inserted into a built-in fluorescence or brightfield illumination path of a widefield microscope to create a pattern onto a specimen in order to improve resolution. Generally, this requires inserting the grid or diaphragm into a field diaphragm slot of the microscope. However, this method becomes dependent on manipulating the existing built-in light path of the microscope. The built-in optical paths in some conventional microscopes were generally not designed to project such patterns onto the object plane accurately with high contrast and without distortion of the patterns. The manipulation of the built-in light path may also diminish the efficiency of the built-in fluorescence source.
Some conventional widefield microscopes have also not been designed to optimize the Point Spread Function of a point object. The Point Spread Function is a measure of the quality of the optical device. In incoherent imaging systems such as fluorescent microscopes the image formation process is linear and described by Linear System theory. Knowing the Point Spread Function of an optical device allows the use of deconvolution algorithms to generate software enhanced images, referred to as deconvolution microscopy. The better the intrinsic Point Spread Function of an optical device, the higher the quality of the image and the less computational complexity would be involved in deconvolving the image. The built-in optical paths in available widefield microscopes generally produce a high degree of out of focus light owing to their architecture, and there is high computational complexity when using deconvolution microscopy techniques.